Cervical cancer is one of the primary causes of death for women worldwide and in Taiwan. According to the statistics by the World Health Organization (WHO) in 2002, cervical cancer is the second cause of death for women cancers worldwide, only subsequent to breast cancer. Periodic cervical cancer screening is the best way of cervical cancer prevention. Currently, there are two methods for cervical cancer screening. One is the most commonly known Pap smear and the other is HPV testing. Pap smear is performed by obtaining the discharge from the cervical, and observing whether cancerous lesion occurs in the detached epithelial cells by microscopy for early-stage detection of cervical cancer. HPV testing is performed by using polymerase chain reaction (RT-PCR) or Hybrid Capture to examine whether human papilloma virus (HPV) is present in the sample.
However, because it requires physicians to obtain the sample and medical technologists/pathologists to interpret the data from of smear, in addition to high false negative rate and the subsequent delay in diagnosis and treatment of the precancerous lesion, the required quality in human resource and cost is overly high. For many developing countries, it is difficult to promote. On the other hand, although HPV testing is highly sensitive, it is also prone to high false positive rate. Not only the patient worries in vain, but also much medical resource wasted in the follow-up examinations of these false positive patients. Therefore, there remains a problem of how to elevate the accuracy and convenience of screening methods of cervical cancer for promoting cervical cancer screening.
Genomic deletions are considered an important factor in tumor formation. For a long time, we are used to the concept that the code of genes relies on the permutation and combination of the four bases. Early as in 1975, Knudson proposed the two-hit theory, pointing out that some mutations or deletions accompanying homologous tumor suppressor genes may cause or are prone to cause cancer. However, other information that affects phenotype may exist in the modified base, 5-methylcytosine. 5-methylcytosine is found to exist in the palindrome sequence 5′-CpG-3′ in mammalian cells. In mammalian cells, besides the regions that are called “CpG islands (CGIs),” most CpG dinucleotide pairs are methylated. CpG islands refer to regions having about 1000 base pairs (1 Kb) that contain large amounts of GC- and CpG-. Usually, they are present around the genes, and are found near the promoters of broadly-expressed genes. Methylation of cytosine occurs after DNA synthesis, which transfers methyl group from the methyl group donor, S-adenosylmethionine (SAM), to the 5th carbon of cytosine. This enzymatic reaction is carried out by DNA methyltransferase (DNMTs). DNMT1 is the primary methyltransferase in mammals, which is responsible for the post-replicative restoration of hemi-methylated positions to full methylation, and thus maintenance of methylation. On the other hand, DNMT3A and DNMT3B are considered to be responsible for methylation of new positions, a process called de novo methylation.
Loss of methylation in CpG dinucleotide pairs refers to the generally-known low degree of methylation which is the first epigenetic abnormality in cancer cells. However, researches in past few years indicate that site-specific hypermethylation (such as in some tumor suppressor genes) is associated with loss of function. This may provide selective advantages during tumor formation. Hypermethylation of CpG islands in the promoter region may induce chromatin remodeling through gene silencing accompanied with histone modification. In addition to chromosome deletion and gene mutation, epigenetic silencing of tumor suppressor genes caused by hypermethylation of promoter is also common in human cancers.
Recent researches in epidemiology demonstrate that the concentration of serum folate (a primary source of methyl group) is associated with the infection and clearance of HPV. In the metabolism of methyl cycle, genetic polymorphisms of enzymes are also reported as associated with the development of cervical epithelial lesions. Like the concept of super gene evolution, researches on the association between DNA methylation and cervical cancer are also prevailing. Researches on DNA methylation of cervical cancer increase with time, indicating the possibility of using methylation for cervical cancer screening. Due to the interaction between genetics and the environment, the degree of methylation of tumor suppressor genes varies among different genes and different populations. Different diseases may have different methylator phenotypes. However, the methylator phenotype of cervical cancer and association with HPV are still unknown. What specific genes would be methylated in cervical cancer and how many genes is required to meet the need in clinical application remain the issues that need to be verified in the future.
Based on the above, the current methods of cervical cancer screening still have many defects and are not properly designed. An improvement is needed.
The inventors of the subject application have filed relevant patent applications in Taiwan (TW Pat. Pub. No. 200831900, TW Pat. Pyb. No. 201038739), China (CN Appl. No. 200810094659.2, CN Appl. No. 200910135501.X), Malaysia (UI20085354) and the USA (US Pat. Pub. No. 20080311570, US Pat. Pub. No. 20110045465) (hereafter refers to as the prior applications). The method III for screening cancer is an extension of the prior applications. The inventors of the subject application discovered novel biomarkers for cancer screening and the screening methods.